Biomedical subjects
Publications and source records attributed to C C Gibson.
Toward the end of creating educational programs that provide both success in and access to learning, continuing medical education is challenged to consider new perspectives on learning and the incorporation of instructional technologies for teaching.
The diffusion coefficients of two caged fluorescent dyes were measured in free solution and in the lateral intercellular spaces (LIS) of cultured Madin-Darby canine kidney (MDCK) cells after photoactivation by illumination with a continuous or pulsed UV laser. Both quantitative video imaging and a new photometric method were utilized to determine the rates of diffusion of the caged fluorescent dyes: 8-((4,5-dimethoxy-2-nitrobenzyl)oxy)pyrene-1,3,6-trisulfonic acid (DMNB-HPTS) and (4,5-dimethoxy-2-nitrobenzyl) fluorescein dextran (10,000 MW) (DMNB-caged fluorescein dextran). The diffusion coefficients at 37 degrees C in free solution were 3.3 x 10(-6) cm2/s (HPTS) and 0.98 x 10(-6) cm2/s (10,000 MW dextran). Diffusion of HPTS within nominally linear stretches of the LIS of MDCK cells grown on glass coverslips was indistinguishable from that in free solution, whereas dextran showed a 1.6 +/- 0.5-fold reduction in diffusivity. Measurements of HPTS diffusion within the LIS of multicellular regions also exhibited a diffusivity comparable to the free solution value. The restriction to diffusion of the dextran within the LIS may be due to molecular hindrance.
Explore the source record for details and available documents.
Recent studies in a variety of cell types have revealed several receptor subtypes that bind ATP and trigger increases in intracellular Ca2+ concentration ([Ca2+]i). The present studies were aimed at determining whether similar receptors are present in the rat terminal inner medullary collecting duct (IMCD). [Ca2+]i was measured using fura 2 in tubules dissected from collagenase-treated rat kidneys. ATP (1-100 microM) caused a rapid increase in [Ca2+]i with a prolonged late phase after an initial peak. A similar rise was observed in tubules exposed to UTP or to the poorly hydrolyzable analogue, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). In contrast, agonists that bind P2x, P2y, P2z, and P2t purinergic receptors did not affect [Ca2+]i. Removal of extracellular Ca2+ inhibited the response to ATP by approximately 50% with obliteration of the late phase. Furthermore, indomethacin attenuated the rise in [Ca2+]i produced by ATP. Adenosine analogues also increased [Ca2]i apparently by binding to distinct adenosine receptors rather than to the ATP receptor. We conclude that there is a nucleotide receptor in the rat terminal IMCD, which, when occupied, mobilizes intracellular Ca2+.
Explore the source record for details and available documents.
In renal collecting duct epithelial cells, arginine vasopressin (AVP) at greater than nanomolar concentrations has been reported to transiently increase intracellular free calcium ([Ca2+]i) in a manner consistent with activation of the phosphoinositide pathway. To investigate whether any of the known neurohypophysial hormone subtypes are involved, we measured [Ca2+]i in microdissected rat terminal inner medullary collecting duct (IMCD) using fura-2. To allow quantitative comparisons of the response under different conditions, we determined the areas under the response curves (in nM.min) over 1.5 min using numerical integration. AVP, the V1b-receptor agonist [deamino1,D-3-(pyridyl)Ala2,Arg8]vasopressin, the V2-receptor agonist 1-desamino-8-D-arginine vasopressin, oxytocin, and the selective oxytocin-receptor agonist [Thr4,Gly7]oxytocin (TG-OXT), each at 10 nM, significantly increased [Ca2+]i (69.52 +/- 10.25, 27.0 +/- 11.7, 24.33 +/- 5.83, 14.75 +/- 2.81, and 14.57 +/- 3.50 nM.min, respectively). In contrast, a V1a-selective agonist ([Phe2,Ile3,Orn8]vasopressin) did not increase [Ca2+]i (0.43 +/- 2.36 nM.min). In desensitization studies, challenge with 10 nM AVP or TG-OXT completely prevented a rise in [Ca2+]i in response to immediate rechallenge with the same agent, but not the other, demonstrating homologous desensitization. The lack of cross-desensitization implies that at least two receptors are present that can trigger a rise in [Ca2+]i in response to neurohypophysial hormones. Antagonists for oxytocin ([des-glycinamide9,d(CH2)5(1),O-Me-Tyr2,Thr4,Orn8]vaso tocin), V2 ([d(CH2)5(1),D-Ile2,Ile4,Arg8]vasopressin), and V1a ([d(CH2)5(1),O-Me-Tyr2,Arg8]vasopressin) receptors partially inhibited the [Ca2+]i response induced by 10 nM AVP (89.5, 81.6, and 51.4% inhibition, respectively). These data are consistent with the view that both an oxytocin receptor and a vasopressin receptor are coupled to a [Ca2+]i mobilization response in rat terminal IMCD. This vasopressin receptor is distinct from both the V1a receptor and the V2 receptor and may be either the V1b receptor or a novel vasopressin receptor subtype.
We have developed a new application of the fluorescence photobleaching recovery (FPR) technique for instantaneous measurement of volume flow rates at any axial position along isolated perfused kidney tubules. The method requires fast data acquisition of emitted fluorescence through a photomultiplier (time resolution, 0.5 ms) coupled with differential interference contrast microscopy to measure luminal diameters accurately. While the tubule is perfused in vitro with an impermeant fluorophore (fluorescein sulfonate), a 20-ms bleach pulse reduces the fluorescence in the observation region by 20-25%. Fluorescence recovery is a direct function of perfusate velocity; diffusion plays no significant role in the early phase of recovery. A fluid dynamics approach to data analysis shows that fractional recovery increases linearly with time until t = L/2vm, where L is the length of the observation window and vm is the mean axial velocity. Practically, a linear regression analysis of the early recovery phase allows measurement of vm of up to 0.14 cm/s, i.e., a 40-nl/min flow rate in a 25-microns-diameter tubule. Calibration experiments in small glass tubes perfused at predetermined flow rates demonstrated good accuracy (within 10%) and reproducibility (coefficient of variation, 8.7%). In rat inner medullary collecting ducts microperfused at 4-40 nl/min, the correlation with a standard fluid collection method was excellent (r2 greater than 0.97). The method should also be suitable for the direct measurement of fluid flow rate in kidney tubules or blood vessels microperfused in vivo.
The glomeruli of mice transgenic for bovine growth hormone (GH mice) were disproportionately enlarged as a function of either kidney or body weight. Glomerular size correlated with mesangial sclerosis and the urine albumin/creatinine ratio. The glomerular lesions consisted of mesangial proliferation (4 to 5 weeks) followed by progressive mesangial sclerosis (19 weeks), resulting in complete glomerulosclerosis at 30 to 37 weeks. Albuminuria paralleled the glomerulosclerosis. In contrast, mice transgenic for insulinlike growth factor-I (IGF-I mice) did not develop glomerulosclerosis, even though glomerular size significantly increased. Glomerular hypertrophy, however, did not reach that in GH mice. These data suggest that high levels of circulating GH lead to a disproportionate increase in glomerular cellularity and volume, as well as glomerulosclerosis. This does not appear to be the result of high levels of circulating IGF-I stimulated by GH, as the serum IGF-I level in GH mice was lower than that in IGF-I mice.
Previous studies of the erythropoietic response to hypoxia in high-altitude natives suggest that the hematocrit and hemoglobin values in Himalayan natives (Sherpas) are lower than expected for the altitude, perhaps because of a genetic adaptation. However, differences in sampling techniques and experimental methods make comparisons difficult. Our studies were carried out to compare the erythropoietic response with the same altitude in age-matched natives of the Himalayas and Andes by the same experimental techniques. Healthy male subjects were selected in Ollagüe, Chile (n = 29, 27.3 +/- 5.9 yr) and in Khunde, Nepal (n = 30, 24.7 +/- 3.8 yr). Both of these villages are located at 3,700 m above sea level. Hematologic measurements confirmed lower hematocrit values in Nepal (48.4 +/- 4.5%) than in Chile (52.2 +/- 4.6%) (P less than 0.003). When subjects were matched for hematocrit, erythropoietin concentrations in Chile were higher than in Nepal (P less than 0.01). Detailed measurements of blood O2 affinity in Nepal showed no differences in shape or position of the O2 equilibrium curve between Sherpas and Western sojourners. Our results indicate that although Quechua Indians have higher hematocrits than Sherpas living at the same altitude, nevertheless they may be functionally anemic.
The total DNA per cell and DNA synthetic cycle phases were determined by flow cytometry in five Candida isolates including three species: Candida albicans 208R1, Candida tropicalis ATCC 750, and Candida parapsilosis 970, 3138, and ATCC 22019. The cells were prepared for flow cytometry by fixation in Carnoy fixative followed by staining with mithramycin. Marked but stable and reproducible inter- and intraspecific differences in total DNA per cell of stationary-phase cultures were found which did not correlate directly to diphenylamine estimates of the same parameter. This discrepancy was resolved by mathematically converting flow cytometry data into diphenylamine data. The reason for the discrepancy was found in studies of the DNA synthetic cycle of these yeasts: a large but isolate-specific variable proportion of the population is arrested in the S and G2-M phases after the culture passes from exponential to stationary phase. Histograms of exponential-growth-phase Candida isolates demonstrate that the majority of the population is in the G2-M phase of the DNA synthetic cycle. The DNA content of the C. tropicalis and C. parapsilosis isolates studied is as high as or higher than that of C. albicans. Extranuclear fluorescent particles were observed in the C. tropicalis isolate. No equivalent particles could be detected in the other four Candida isolates. The nature of the particles is unknown.
We have employed both small unilamellar vesicles (SUV) and large unilamellar vesicles formed by the reverse phase evaporation technique (REV) to study the initial kinetics of membrane aggregation and fusion. Stopped flow measurements of the calcium-induced changes in the turbidity of SUV and REV, formed from 1:1 (mol/mol) mixtures of bovine phosphatidylserine (PS) and Escherichia coli phosphatidylethanolamine (PE), were used to follow particle aggregation. Simultaneous measurements of the fluorescence resonance energy transfer from N-(7-nitro2,1,3-benzoxadiazol-4-yl) (NBD)-PE to rhodamine (Rho)-PE incorporated into the vesicle bilayers established that 1) both initial aggregation and fusion can be described as a bimolecular process and 2) the rate-limiting step of membrane fusion is aggregation. Thus fusion takes place in the microsecond time domain. Parallel experiments, which simultaneously measured aggregation and the dequenching of encapsulated carboxyfluorescein (CF) in the presence and absence of antifluorescein antibodies in the suspension medium, established that the small unilamellar vesicles rapidly lose their contents of CF as they fuse. On the other hand, the first few cycles of fusion of the large unilamellar vesicles are nonleaky, but leakage develops within 1-2 s as the particles grow in size. Thus the results demonstrate that the SUV are poor models for the study of nonleaky fusion, while the REV must be carefully tested before unambiguous interpretation of fusion assays involving the formation of tight complexes (such as the terbium-dipicolinic assay) can be made. NBD-PE undergoes very rapid, Ca2+-promoted changes in quantum yield which can obscure the resonance energy transfer signals. Thus data from the NBD-PE/Rho-PE energy transfer pair must be carefully scrutinized for artifacts.
Oxygen equilibrium curves of 48 healthy adult subjects have been measured by the method of Rossi-Bernardi et al. (Clin. Chem. 21: 1747, 1975), in which H2O2 is gradually added to a sample of deoxygenated blood that contains an excess of catalase. The mean P50 for nonsmokers was 26.9 Torr and the distribution of values was slightly skewed to the right (range 24.2--29.9 Torr). The method differs from others previously available in that pH, PCO2, and HCO3-- are constant during oxygenation. The system for control of the experiment and data collection and processing has been automated by the use of a microprocessor so that the equilibrium curve can be obtained quickly, reproducibly, and relatively simply. With the aid of a digital computer, the parameters of the generalized Adair equation can also be estimated.
Sixteen RN-to-BSN students participated in interviews at the start and at the completion of taking their first class from a newly implemented, university-based, collaborative distance learning program. The purpose of the taped, semi-structured telephone interviews was to identify the support RNs experienced. Institutional themes of support that emerged were orientation programs, technical support, mentors, library resources, and prompt feedback from instructors. Non-institutional support themes focused on family, friends, peers, the workplace, and learners.